1. Field of the Invention
The invention generally relates to the use of a targeting fragment of a toxin or lectin molecule for the delivery of a substance of interest to cells. In particular, the invention provides a composition comprising a targeting fragment of a toxin molecule and a substance of interest, and methods for use of the composition. More particularly, the substance of interest may be a photosensitizing agent for use in targeted cell killing, or a visualizing agent for use in identifying cell surface receptors of interest.
2. Description of Related Art
The xe2x80x9choly grailxe2x80x9d of research in the battle against cancer has been the development of a magic bullet to selectively kill cancerous cells while leaving normal cells untouched. Standard, therapeutic approaches to the treatment of cancer include surgery to remove the cancerous tissue (if the tumor is well defined and localized), radiotherapy, chemotherapy or combinations of these methods. Frequently, when a cancerous tumor is removed, a significant portion of surrounding tissue is also removed to ensure that the majority of cancerous cells are eliminated. In some cases, an entire organ is removed, even though portions of the organ are still healthy. In spite of such radical procedures, cancer cells may spill into body cavities and remain behind to proliferate. Further, portions of a tumor may be difficult to discern or difficult to access. The ability to accurately target cancerous cells for destruction while leaving normal, healthy tissue intact would be a major step forward in the treatment of this disease.
One form of therapy that is currently gaining acceptance for the treatment of hyperproliferating tissues is photodynamic therapy (PDT). Based on the discovery made over years ago that rapidly growing cells treated with certain chemicals will die when exposed to light, PDT is currently being used to treat several different types of cancers and non-malignant lesions. There appears to be a selective affinity and retention of photosensitizers in hyperproliferating tissue. Commonly, a patient is injected with a photosensitizer (PS) molecule that spreads throughout the body. There is then a waiting period during which the PS molecules accumulate in the target tissue, and are eliminated from most non-target tissue. Light is then used to illuminate a mass of tumor cells and activate the PS molecules to produce singlet oxygen, thereby killing cells and tissue in the area. The use of PDT to treat esophageal cancer, lung cancer and macular degeneration is currently being evaluated in clinical trials. One of the theoretical advantages of PDT is that tissues unexposed to light will not be affected. However, in reality, the rate of clearance of the photosensitizer from normal tissue is highly variable. Thus, while success rates of treatment with PDT are so far impressive, deleterious side effects such as skin sensitivity to light for four to six weeks have been observed. In addition, inflammation of the treatment site resulting in shortness of breath and coughing has been observed as a result of PDT treatment of lung and esophageal cancers.
Attempts have been made to optimize PDT treatments. U.S. Pat. No. 6,058,937 to Dorion et al., the disclosure of which is incorporated herein by reference, presents a method for shortening the waiting period after administration of PDT to a tissue. The technique is limited, however, to highly vascularized tissue. Rather than destroying the tissue itself, PDT is used to destroy the vasculature that nourishes the tissue and thus indirectly causes tissue death. The majority of molecules do not readily penetrate cell membranes. Methods for introducing molecules of interest into the cytosol of living cells are disclosed in U.S. Pat. No. 5,876,989 to Berg et al., the disclosure of which is incorporated herein by reference. The molecules to be released into the cell""s cytosol are first taken up in endosomes, lysosomes or other cell compartments together with a photosensitizing compound. Light activation of the photosensitizing compound is then used to rupture the membranes of the cell compartments. The contents of the ruptured compartments (including the molecule of interest) are released into the cell cytosol without killing the majority of the cells. This invention thus utilizes PDT as a mechanism for releasing a drug (such as gelonin, a ribosome inactivating protein) from endosomes/lysomes to the cell compartment where the drug is effective (cytosol). The method does not provide a method of cell killing by PDT.
Kraus et al. in U.S. Pat. No. 6,160,024 describe chemical linkers to connect an energy emitting compound to a photosensitizing molecule, thereby providing an internal chemically-activated light source for activation of the photosensitizer. The method was employed to destroy virus-infected or tumor cells. However, this and other known PDT methods generally lack specificity. Their use results in whole body sensitization to illumination and the attendant side effects.
A need, therefore, exists for a way to enhance the specificity of PDT in order to avoid whole-body light sensitivity.
In order to enhance the specificity of cancer therapies, researchers have attempted to take advantage of the many biochemical and physiological changes that occur during cancer cell transformation. Some of these changes include the presence of cell-surface molecules when cells become cancerous. This has led to the use of antibodies coupled with toxic compounds to selectively bind to the surface of cancerous cells thereby killing those cells. However, this approach also has limitations that include the heterogeneous uptake of the toxin by the tumor cells, the slow elimination of the antibody-toxin complex from the blood system, and the cross-reactivity of the antibody with normal tissue.
The differential expression of many cell-surface molecules in human cancerous cells has been well studied and thoroughly documented. One such molecule is globotriaosylceramide (also known as Gb3, CD77 and pk antigen). The Gb3 glycosphingolipid is normally expressed in several tissues including intestinal epithelium, kidney epithelium, and endothelial cells, in addition to being found in human milk as a glycolipid. Gb3 is also expressed in a fraction of germinal center B lymphocytes, and traces of Gb3 are found in red blood cell membranes of most individuals. Gb3 is strongly expressed in the red blood cell membranes of pk blood type individuals (0.01% of the population). The over-expression of this cell-surface receptor has been documented in ovarian cancer, Burkitt""s lymphoma (non-Hodgkin""s lymphoma), breast cancer, brain cancer, gastric cancer, and testicular cancer. It would not be unreasonable to predict that the over-expression of Gb3 may occur in many other types of cancer, as well.
The Gb3 receptor (intestinal) is targeted by the bacterial toxin proteins belonging to the verotoxin family of bacteriotoxins that includes the Shiga toxins and Shiga-like toxins. Bacterial (Shigella dysenteriae and Escherichia coli) production of these toxins leads to disorders such as food poisoning, dysentery, hemorrhagic colitis, and hemolytic uremic syndrome. It is not the actual bacterial infection, but the production of the toxin molecules that leads to the disease symptoms. The bacteriotoxins, both Shiga and Shiga-like, are comprised of two protein components, a catalytic A subunit and a pentameric B subunit. The catalytic A subunit is a potent N-glycosidase that inhibits protein synthesis once inside a cell. The B subunit array is responsible for targeting specific cells expressing Gb3 on their surface by recognizing and binding the Gb3 receptor. Several issued patents take advantage of the targeted specificity of verotoxins to localized positions on cancerous cells.
Verotoxin 1, or the pentameric B subunit of verotoxin 1, administered in a non-lethal amount has been effective in treating mammalian neoplasia, including ovarian, brain, breast, and skin cancers as described, for example, in Lingwood et al., U.S. Pat. No. 5,968,894, the disclosure of which is incorporated herein by reference.
Gariepy in U.S. Pat. No. 5,801,145, the disclosure of which is incorporated herein by reference, describes the treatment of non-Hodgkin""s lymphomas by a related ex vivo method. Taking advantage of the fact that Shiga toxin or Shiga-like toxin-1 selectively binds to CD77+ cells, according to this method bone marrow cells are treated with the toxins to specifically kill CD77+ cells prior to a bone marrow transplant.
While some of these methods utilize cell targeting via specific cell surface receptors, they lack the benefits of PDT.
An indispensable component of cancer treatment protocols is the ability to detect and locate, i.e. to visualize tumors. The noninvasive visualization of cancerous tumors is technically challenging and has led to the development and application of numerous modalities. These include X-ray, CT scan, nuclear scan, PET scan, MRI, ultrasound and numerous modifications that are used to visualize tumors. These techniques are used routinely for general biomedical imaging with each having distinct advantages and disadvantages. Overall, the most significant disadvantage of these techniques is the lack of sensitivity to detect small abnormalities. As such, a high level of skilled expertise is required by personnel to identify a small cancerous tumor. The detection and identification of a cancerous growth is paramount to achieving a positive outcome for the patient. The ability to readily distinguish between cancerous cells and normal cells would be of tremendous benefit to an oncologist or clinician.
Thus, there is also an existing need for a method that allows specific detection, localization, and visualization of cancerous cells. A methodology that provides noninvasive imaging of tumors and simple visual discrimination of cancerous versus normal cells would be highly beneficial.
Therefore, the principle need is for a vehicle and method for delivery of substances of interest specifically to targeted cells that provides all of the above referenced advantages without the disadvantages associated with each taken individually.
It is a principle objective of the present invention to provide a methodology of delivering molecules of interest to cells. The method involves associating a substance of interest with a targeting fragment of a toxin or lectin molecule. The targeting fragment mediates the cellular delivery and internalization of the substance of interest. The substance of interest will be internalized by cells possessing the cell surface receptor for which the targeting fragment is specific.
In one aspect, the present invention targets a cell surface receptor (CD77, Gb3) identified as being over expressed in ovarian cancer cells, Burkitt""s lymphoma cells, breast cancer cells, gastric cancer cells, and testicular cancer cells. The receptor is naturally expressed in intestinal epithelium, kidney epithelium and endothelial cells, in addition to being found in human milk. This receptor is naturally targeted by the B subunit of the bacterial protein SLT. Once bound to the receptor via the B subunit, the entire protein is internalized by the cell. The B subunit thus functions to xe2x80x9cdeliverxe2x80x9d the catalytic A subunit of SLT to the cell. By coupling a substance of interest to the B subunit of SLT (SLT-B) in lieu of the A subunit, it is likewise possible to deliver the substance of interest to cells possessing the Gb3 receptor.
In one embodiment of the present invention, the substance of interest is a photosensitizing agent. For example, the photosensitizing agent chlorin e6 (Ce6) has been well studied and used as a model system for photodynamic therapy. The instant invention describes coupling Ce6 with SLT-B to affect cell killing using light.
In one embodiment of the instant invention, the substance of interest that is attached to the targeting fragment and delivered to a target cell is a visualizing agent. The resulting targeting fragment/visualizing agent composition would be useful for identifying cell surface receptors of interest in patients and clinical samples. This application includes the identification of cell surface receptors in both pathogenic (e.g. cancerous) tissue and normal tissue. Examples of suitable clinical samples include but are not limited to biopsy samples, blood samples, bone marrow samples, and the like.
For the practice of this aspect of the invention, a composition comprising a targeting fragment of a toxin or lectin molecule and a visualizing agent are provided to a patient, or to a clinical sample, and the cell surface receptor of interest is then located in the patient or the clinical sample by imaging the visualizing agent after the targeting fragment has bound to the receptor of interest. Those of skill in the art will recognize that many types of imaging exist that could be utilized in the practice of this aspect of the invention, including but not limited to such procedures as scintigraphy (nuclear scanning), x-ray or CT scans, magnetic resonance imaging (MRI), and the like. Any suitable type of imaging may be utilized in the practice of the present invention so long as the visualizing agent that is delivered via the targeting fragment can be detected.
In one embodiment of the present invention, the cell surface receptors that are identified by the practice of the present invention are associated with cancer cells and identification of the cell surface receptors allows visualization of the cancer cells. However, those of skill in the art will recognize that the visualization of cells displaying such receptors would be advantageous in many circumstances. Examples include but are not limited to the visualization of tissues (normal or diseased) prior to or during surgical procedures, localization of specific cell types, monitoring cell location, and the like. The method of the present invention may be utilized to identify any cell surface receptor of interest, so long as the receptor binds with specificity to a targeting fragment of a toxin or lectin molecule.
The practice of this aspect of the present invention would provide a distinct advantage in, for example, identifying and locating tumors or other cancerous cells and thus for planning treatment protocols. The method could aid in the assessment of metastasis, or be useful during surgery to remove tumors. For example, the practice of the method of the present invention would render cancerous tissues visible and distinguishable from normal tissue. It would thus be possible to be more conservative with respect to removal of tissue that is not cancerous, thereby minimizing the loss of healthy tissue by the patient.
It is yet another objective of the present invention to provide a methodology for the direct visualization of cancerous cells by complexing a fluorescent molecule to the B fragment, illuminating with an appropriate light and observing the light emitted. This methodology would be used, for example, during biopsy or surgical procedures.
Another objective of the present invention to provide a predictable method of detecting normal expression patterns in intestinal and other tissues. Because the Gb3 receptor is expressed in tissue other than tumor cells, there would be areas of localization of a conjugate regardless of the presence of tumor cells. However, the localization in intestinal tissue or elsewhere would be predictable based upon known normal expression patterns. This embodiment of the invention could be used for imaging of normal intestinal, kidney and endothelial tissues (vasculature) for example to detect changes in expression that may result from physiological changes associated with conditions such as disease, pregnancy, administration of drugs, aging, etc.